Study On Iron-based Nanomaterials For Nucleic Acid Delivery And Gene Regulation

Gene regulation refers to the mechanism by which organisms regulate the level of protein products by controlling the processes of DNA transcription and m RNA translation.RNA interference(RNAi)is an important gene regulation method.This technology introduces the double-stranded RNA(ds RNA)formed by the sense and antisense strand corresponding to the target m RNA sequence into the cell,so that the m RNA is specifically degraded or inhibited,and the corresponding gene is silenced.And this method shows great potential in biomedical research and applications.However,the development and application of RNAi technology are restricted due to poor cellular uptake efficiency and easy degradation by ribonucleases of RNA molecules.Therefore,it is of great significance to develop safe and effective technical means to achieve intracellular delivery of nucleic acid molecules.In recent years,the application of nanomaterials in the field of biomedicine has become more and more extensive,and many materials have been used as nucleic acid nanocarriers with ideal results.In-depth research on the cellular internalization mechanism,intracellular transport pathway and distribution of nanoparticles is helpful for the design,optimization and application of nanocarriers.Live-cell fluorescence imaging is an effective means to study intracellular transport of nanomaterials.Among the numerous nanomaterials,iron-based nanomaterials have attracted much attention due to their superior physicochemical properties,such as the ability to activate the immune system,enhance the Fenton reaction,magnetic hyperthermia,magnetic resonance imaging and so on.In addition,iron-based nanomaterials generally have better biosafety.Therefore,iron-based nanomaterials play an increasingly important role in disease diagnosis and treatment.However,the above applications are all based on the inherent properties of iron-based nanomaterials,and the study of using them as nanocarriers to deliver functional nucleic acids for gene regulation remains to be still under developed.Iron-based nanomaterials can be mainly divided into two categories:iron-based nanocomposites and iron-based nanocrystals.The research objects of this paper include two typical iron-based nanomaterials,namely MIL-101-NH₂(Fe)and Fe₃O₄nanoparticles.MIL-101-NH₂(Fe)is an iron-based nanocomposite material,which is a metal-organic framework material with high porosity,high surface area,high thermal stability,low toxicity and good biodegradability.It has been widely used in the synthesis of composite nanomaterials,chemical sensors,drug delivery and other research fields.Fe₃O₄nanoparticles are iron-based nanocrystals that have been approved by the U.S.Food and Drug Administration(FDA)as contrast agents and drug carriers for magnetic resonance imaging.So Fe₃O₄nanoparticles have excellent biosafety.At present,the application of the above two iron-based nanomaterials as nucleic acid nanocarriers has still to be developed,especially the research on their intracellular transport process is rarely reported.Based on this,this paper studies the intracellular transport process of these two iron-based nanomaterials and their application of delivering functional nucleic acids for gene regulation.It mainly includes the following two parts:1.Intracellular imaging and nucleic acid delivery of MIL-101-NH₂(Fe)Using ferric chloride hexahydrate and 2-aminoterephthalic acid as raw materials,MIL-101-NH₂(Fe)nanoparticles with uniform particle size were successfully synthesized by hot solvent method.Benefiting from the electrostatic interaction between the positive charge of MIL-101-NH₂(Fe)nanoparticles and the negative charge of nucleic acid molecules,the two can be efficiently assembled.It was characterized by DLS,Zeta potential and agarose gel electrophoresis,and the results showed that the nanocomplex(MIL-101-NH₂-NA)of MIL-101-NH₂(Fe)nanoparticles loaded with nucleic acid moleculeswas successfully prepared.The results of cell experiments showed that MIL-101-NH₂(Fe)nanoparticle is a kind of nanocarrier with good biocompatibility and the cellular uptake is concentration-dependent.The results of cell imaging showed that the co-localization of MIL-101-NH₂-NA with lysosomes increased first and then decreased with time,proving that it was degraded in an acidic environment,successfully releasing nucleic acid molecules,and providing the possibility for functional nucleic acid to play a role.Finally,MIL-101-NH₂(Fe)nanoparticles were loaded with si RNA to knock down the green fluorescent protein(EGFP)gene,and the effect of inhibiting protein expression was visualized by fluorescence imaging.2.Intracellular transport imaging and nucleic acid delivery of Fe₃O₄nanoparticlesUsing EDC as a coupling agent,the aminated fluorescent nucleic acid chain was modified on the carboxylated Fe₃O₄nanoparticles,and the spherical nucleic acid(Fe₃O₄-SNA)with Fe₃O₄as the core was constructed.The experimental results of DLS,Zeta potential and agarose gel electrophoresis proved the successful preparation of Fe₃O₄-SNA,and the loading rate of Fe₃O₄nanoparticles carrying nucleic acid molecules was calculated to be 4.98 nmol/mg.In vitro enzyme activity experiments showed that Fe₃O₄nanoparticles still possessed the dual enzyme activities of peroxidase and catalase after the nucleic acid was modified.In addition,the strand displacement reaction further proved that Fe₃O₄-SNA has a good hybridization ability,which provides a basis for the subsequent gene therapy and detection of functional nucleic acid loaded.The ICP-MS results showed that compared with bare Fe₃O₄nanoparticles,the nucleic acid molecule modification promoted the cellular uptake of Fe₃O₄-SNA,which provided the possibility for the subsequent use of fluorescent DNA-labeled Fe₃O₄-SNA to explore the intracellular transport process of Fe₃O₄nanoparticles.The results of intracellular imaging and real-time tracking showed that Fe₃O₄-SNA entered cells through caveolin-mediated endocytosis,and was transported along microtubules,triggering the formation of autophagosomes,and finally reaching lysosomes.A series of experiments showed that Fe₃O₄-SNA does not affect cell viability,intracellular redox balance and autophagy effect,and is suitable as a nanocarrier for functional nucleic acids.Finally,Fe₃O₄-D/R was prepared by designing a DNA strand complementary to mi RNA-34a,hybridizing it with mi RNA-34a,and modifying the double strand onto Fe₃O₄nanoparticles.The results show that Fe₃O₄-D/R can effectively enter cells.And it can effectively down-regulate the expression level of survivin gene in cells,and can inhibit the growth of cancer cells.The nanocomposite provides a feasible strategy for Fe₃O₄nanoparticles to deliver functional nucleic acids.